Human narcolepsy was recently associated with a decline in orexin/hypocretin containing neurons. These neurons are located only in the lateral hypothalamus, a region not previously implicated in narcolepsy or REM sleep. How could destruction of these cells lead to narcoleptic behavior? One way to answer this question would be to determine whether lesion of the target neurons produces narcoleptic symptoms. However, there are no studies demonstrating which hypocretin/orexin innervation to what target area regulates which aspect of sleep-wake behavior. To target the cells which express the hypocretin/orexin receptors in adult animals, we have conjugated the ribosome inactivating protein, saporin, to hypocretin-2/orexin B. We will utilize the orexin-saporin to test the overall hypothesis that hypocretin/orexin via its innervation of specific targets promotes wakefulness and inhibits REM sleep. Specific aim 1 will test the hypothesis that orexin/hypocretin innervation to the dorsolateral pons regulates REM sleep and cataplexy. Historically, the dorsolateral pontine area has been implicated in regulating REM sleep. Since narcoleptic canines and the orexin gene knockout mice show cataplexy and rapid onset of REM sleep, destruction of the hypocretin/orexin-receptor containing neurons should result in cataplexy and REM sleep. Specific aim 2 will test the hypothesis that the orexin/hypocretin neuronal innervation of the TMN is important for wakefulness. Since narcoleptics are excessively sleepy, it is possible that the hypocretin/orexin influence on wakefulness may come via innervation of neurotransmitter containing populations such as the TMN, LC and basal forebrain. Specific aim 3 will examine the effects of orexin-saporin applied to the basal forebrain on sleep and wakefulness. Besides the TMN, wake-active neurons located in the basal forebrain are also hypothesized to promote wakefulness, and hypocretin/orexin fibers innervate this region. It is also hypothesized that degenerating axon in this region might underlie the emotional triggering of cataplectic attacks in narcolepsy (Siegel et al., 1999). Since application of orexin-saporin destroys orexin-receptor bearing neurons, this is an excellent method that can be used to evaluate the degenerating axon hypothesis. Specific aim 4 will examine the effects of orexin-saporin applications to the VLPO-preoptic area. In the previous cycle we demonstrated that lesions of the sleep-active cells in VLPO produce long-lasting insomnia. Since hypocretin/orexin fibers innervate this area it is necessary to determine whether orexin-saporin administered to the VLPO produces long-lasting hypersomnia. Specific aim 5 will determine the effects of the orexin-saporin on neurons containing the hypocretin-1 versus hypocretin-2 receptors. This will determine which receptor subtype containing neurons are affected by the saporin conjugate. Our findings will provide a framework for integrating the hypocretin/orexin cells within an overall model of sleep regulation.